Improved serum albumin binding immunoglobulin single variable domains

ABSTRACT

The present invention relates to amino acid sequences that can bind to serum albumin. In particular, the present invention relates to immunoglobulin single variable domains, and in particular heavy-chain immunoglobulin single variable domains, that can bind to serum albumin. The invention also relates to proteins, polypeptides and other constructs, compounds, molecules or chemical entities that comprise at least one of the immunoglobulin single variable domains binding to serum albumin that are described herein.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/465,188, filed May 30, 2019, which is a national stage filing under35 U.S.C. § 371 of International Patent Application PCT/EP2017/081818,filed Dec. 7, 2017, which claims priority under 35 U.S.C § 119(e) toU.S. Provisional Application Ser. No. 62/430,972, filed Dec. 7, 2016,the contents of each of which are incorporated herein in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created Jun. 28, 2022, isnamed A084870201US02-SEQ-CRP.txt, and is 170,421 bytes in size.

The present invention relates to amino acid sequences that can bind toserum albumin.

In particular, the present invention relates to immunoglobulin singlevariable domains, and in particular heavy-chain immunoglobulin singlevariable domains, that can bind to serum albumin.

As described herein, the immunoglobulin single variable domains providedby the invention are preferably such that they can (at least) bind (andin particular, specifically bind) to human serum albumin. Morepreferably, as further described herein, these immunoglobulin singlevariable domains are preferably further such that they arecross-reactive (as described herein) between human serum albumin andserum albumin from at least one other species of mammal.

The invention also relates to proteins, polypeptides and otherconstructs, compounds, molecules or chemical entities that comprise atleast one of the immunoglobulin single variable domains binding to serumalbumin that are described herein.

Immunoglobulin single variable domains will also generally be referredto herein by means of the abbreviations “ISV's” or “ISVD's” (which willbe used interchangeably herein).

The immunoglobulin single variable domains binding to serum albumin thatare described herein will also be referred to herein as “amino acidsequences of the invention”, or “serum albumin binders of theinvention”. As further described herein, the albumin binders of theinvention may in particular be Nanobodies (as further described herein).

The proteins, polypeptides and other constructs, compounds, molecules orchemical entities that comprise at least one of the serum albumin binderof the invention will also referred to herein as “compounds of theinvention” or as “polypeptides of the invention”. Preferably, thecompounds of the invention are proteins or polypeptides, and may inparticular be fusion proteins.

Other aspects, embodiments, features, uses and advantages of theinvention will be clear to the skilled person based on the disclosureherein.

In the present application, the amino acid residues/positions in animmunoglobulin heavy-chain variable domain will be indicated with thenumbering according to Kabat. For the sake of convenience, FIG. 1 givesa table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT. Note: unlessexplicitly indicated otherwise, for the present description and claims,Kabat numbering is decisive; other numbering systems are given forreference only).

With regard to the CDR's, as is well-known in the art, there aremultiple conventions to define and describe the CDR's of a VH or VHHfragment, such as the Kabat definition (which is based on sequencevariability and is the most commonly used) and the Chothia definition(which is based on the location of the structural loop regions).Reference is for example made to the website bioinf.org.uk/abs/. For thepurposes of the present specification and claims, even though the CDRsaccording to Kabat may also be mentioned, the CDRs are most preferablydefined on the basis of the Abm definition (which is based on OxfordMolecular's AbM antibody modelling software), as this is considered tobe an optimal compromise between the Kabat and Chothia definitions.Reference is again made to the website bioinf.org.uk/abs/).

Accordingly, in the present specification and claims, all CDRs aredefined according to the Abm convention, unless explicitly statedotherwise herein.

ISVD's (and in particular Nanobodies) that can bind to serum albumin andtheir uses are well-known in the art, for example from WO 2004/041865,WO 2006/122787, WO 2012/175400, WO 2015/173325 and PCT/EP2016/077973,which describe serum albumin-binding ISVD's and their use for extendingthe serum half-life (as defined in these applications) of therapeuticcompounds, moieties and entities. For example, WO 2006/122787 disclosesas SEQ ID NO: 62 a humanized serum albumin-binding Nanobody called Alb-8(see SEQ ID NO:1 herein). WO 2012/175400 discloses as SEQ ID NO: 6 ahumanized serum albumin-binding Nanobody called Alb-23D (see SEQ ID NO:2herein). The amino acid sequences of Alb-8 and Alb-23D and their CDR's(which are the same for Alb-8 and Alb-23D) are given in Table A below asSEQ ID NO: 1, 2 and 3 to 8, respectively.

Some other references that disclose ISVD's against serum albumin includeWO 2003/035694, WO 2004/003019, EP 2 139 918, WO 2011/006915 and WO2014/111550.

FIGS. 3A and 3B show alignments of Alb-8, Alb-23D, SEQ ID NO: 15 and thereference albumin binders of SEQ ID NOs: 79 and 80 (which are based onAlb-8 and Alb23, respectively).

The present invention aims to provide improved serum albumin binders,and in particular serum albumin binders that have improved propertiescompared to the serum albumin binders known in the art.

TABLE A Alb-8, Alb-23D and their CDRs SEQ ID NO Description Sequence 1Alb-8 EVQLVESGGGLVQPGNS (WO 2006/ LRLSCAASGFTFSSFGM 122787;SWVRQAPGKGLEWVSSI SEQ ID SGSGSDTLYADSVKGRF NO: 62) TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSL SRSSQGTLVTVSS 2 Alb-23D EVQLLESGGGLVQPGGS (WO 2012/LRLSCAASGFTFRSFGM 175400; SWVRQAPGKGPEWVSSI SEQ ID SGSGSDTLYADSVKGRFNO: 6) TISRDNSKNTLYLQMNS LRPEDTAVYYCTIGGSL SRSSQGTLVTVSSA 3 CDR1 (Kabat)SFGMS 4 CDR2 (Kabat) SISGSGSDTLYADSVKG 5 CDR3 GGSLSR (Kabat/Abm) 6CDR1 (Abm) GFTFRSFGMS 7 CDR2 (Abm) SISGSGSDTL 8 CDR3 GGSLSR (Kabat/Abm)Note: SEQ ID NOs: 1 and 2 share the same CDRs according to Kabat.However, if the CDRs are defined under the Abm convention, SEQ ID NO: 1has a different CDR1 from SEQ ID NOs: 2 compared to SEQ ID NOs: 2, SEQID NO: 1 has an S at position 30 instead of an R. SEQ ID NO: 5 and SEQID NO: 8 are identical. all CDRs are defined according to the Abmconvention, unless indicated otherwise.

TABLE B SEQ ID NO: 15 and its CDR's SEQ ID NO Description Sequence 15T0235005D04 EVQLVESGGGLVQAGGSLRL (wild type) SCAASGLTFSSYAMGWFRQAPGKERERVVSISRGGGYTYY ADSVKGRFTISRDNAENTVY LQMNSLKPEDTAVYYCAAARYWATGSEYEFDYWGQGTLVT VSS 9 CDR1 (Kabat) SYAMG 10 CDR2 (Kabat)SISRGGGYTYYADSVKG 11 CDR3 ARYWATGSEYEFDY (Kabat/Abm) 12 CDR1 (Abm)GLTFSSYAMG 13 CDR2 (Abm) SISRGGGYTY 14 CDR3 ARYWATGSEYEFDY (Kabat/Abm)Note: SEQ ID NO: 11 and SEQ ID NO: 14 are identical. all CDRs aredefined according to the Abm convention, unless indicated otherwise.

Generally, the serum albumin-binding ISVD's provided by the presentinvention are variants of the sequence of SEQ ID NO: 15, in that:

-   -   they have the same CDRs (or essentially the same CDR's) as the        sequence of SEQ ID NO: 15; and    -   they have a certain degree of sequence identity with the        sequence of SEQ ID NO: 15 (which degree of sequence identity is        as further described herein).

In particular, serum albumin-binding ISVD's provided by the presentinvention will generally have a (limited) number of “amino aciddifferences” (as described herein) compared to the sequence of SEQ IDNO: 15. These amino acid differences may be present in the CDR's (aslong as the resulting amino acid sequences as such that they retain thefurther properties of the amino acid sequences of the invention that areset out herein) and/or be present in the framework regions, and may inparticular be present in the framework regions (as defined according toKabat and/or according to Abm). For example and without limitation,these amino acid differences may for example be humanizingsubstitutions, substitutions that improve expression in a desired hostcell or host organism, substitutions that improve stability and/orresistance to degradation and/or proteases, mutations that reducebinding by pre-existing antibodies, and/or other mutations that areintended to optimize the sequence of the amino acid sequences of theinvention; or any suitable combination of such amino acid differences.Reference is made to the further disclosure herein.

In a first aspect, the invention relates to an ISVD that can bind (andin particular, specifically bind) to human serum albumin, and that has:

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        SYAMG (SEQ ID NO: 9) or an amino acid sequence that has 2 or 1        amino acid difference(s) (as defined herein) with the amino acid        sequence of SEQ ID NO: 9; and    -   a CDR2 (according to Kabat) that is the amino acid sequence        SISRGGGYTYYADSVKG (SEQ ID NO: 10) or an amino acid sequence that        has 3, 2 or 1 amino acid difference(s) (as defined herein) with        the amino acid sequence of SEQ ID NO: 10; and    -   a CDR3 (according to Kabat) that is the amino acid sequence        ARYWATGSEYEFDY (SEQ ID NO: 11) or an amino acid sequence that        has 3, 2 or 1 amino acid difference(s) (as defined herein) with        the amino acid sequence of SEQ ID NO: 10.

In particular, a serum albumin binder according to this aspect of theinvention may be (and preferably is) as further described herein.

In a more specific aspect, the invention relates to an ISVD that canbind (and in particular, specifically bind) to human serum albumin, andthat has:

-   -   a CDR1 (according to Kabat) that is the amino acid sequence        SYAMG (SEQ ID NO: 9); and    -   a CDR2 (according to Kabat) that is the amino acid sequence        SISRGGGYTYYADSVKG (SEQ ID NO: 10); and    -   a CDR3 (according to Kabat) that is the amino acid sequence        ARYWATGSEYEFDY (SEQ ID NO: 11).

Again, a serum albumin binder according to this aspect of the inventionmay be (and preferably is) as further described herein.

In another aspect, the invention relates to an ISVD that can bind (andin particular, specifically bind) to human serum albumin, and that has:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GLTFSSYAMG (SEQ ID NO: 12) or an amino acid sequence that has 2        or 1 amino acid difference(s) (as defined herein) with the amino        acid sequence of SEQ ID NO: 12; and    -   a CDR2 (according to Abm) that is the amino acid sequence        SISRGGGYTY (SEQ ID NO: 13) or an amino acid sequence that has 3,        2 or 1 amino acid difference(s) (as defined herein) with the        amino acid sequence of SEQ ID NO: 13; and    -   a CDR3 (according to Abm) that is the amino acid sequence        ARYWATGSEYEFDY (SEQ ID NO: 14) or an amino acid sequence that        has 3, 2 or 1 amino acid difference(s) (as defined herein) with        the amino acid sequence of SEQ ID NO: 14.

In particular, a serum albumin binder according to this aspect of theinvention may be (and preferably is) as further described herein.

In a more specific aspect, the invention relates to an ISVD that canbind (and in particular, specifically bind) to human serum albumin, andthat has:

-   -   a CDR1 (according to Abm) that is the amino acid sequence        GLTFSSYAMG (SEQ ID NO: 12) or an amino acid sequence that has 2        or 1 amino acid difference(s) (as defined herein) with the amino        acid sequence of SEQ ID NO: 12; and    -   a CDR2 (according to Abm) that is the amino acid sequence        SISRGGGYTY (SEQ ID NO: 13) or an amino acid sequence that has 3,        2 or 1 amino acid difference(s) (as defined herein) with the        amino acid sequence of SEQ ID NO: 13; and    -   a CDR3 (according to Abm) that is the amino acid sequence        ARYWATGSEYEFDY (SEQ ID NO: 14) or an amino acid sequence that        has 3, 2 or 1 amino acid difference(s) (as defined herein) with        the amino acid sequence of SEQ ID NO: 14.

Again, a serum albumin binder according to this aspect of the inventionmay be (and preferably is) as further described herein.

Generally, the serum albumin binders according to the different aspectsof the invention are preferably such that they have:

-   -   a degree of sequence identity with the sequence of SEQ ID NO: 15        (in which the CDR's and any C-terminal extension that may be        present are not taken into account for determining the degree of        sequence identity) of at least 85%, preferably at least 90%,        more preferably at least 95%;        and/or such that they have:    -   and/or have no more than 7, preferably no more than 5, such as        only 3, 2 or 1 “amino acid differences” (as defined herein, and        not taking into account the CDRs and any C-terminal extension        that may be present) with the sequence of SEQ ID NO: 15.

The serum albumin binders according to the different aspects of theinvention are generally preferably such that they bind to human serumalbumin with a dissociation constant (KD) of 10⁻⁵ to 10⁻¹² moles/literor less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and morepreferably 10⁻⁸ to 10⁻¹² moles/liter, and/or with a binding affinity ofat least 10⁷ M⁻¹, preferably at least 10⁸ M⁻¹, more preferably at least10⁹ M⁻¹, such as at least 10¹² M⁻¹, as determined using ProteOn(reference is made to Example 1). Preferably, a serum albumin binder ofthe invention will bind to the desired antigen with an affinity lessthan 500 nM, preferably less than 200 nM, more preferably less than 10nM, such as less than 500 pM, again as determined using ProteOn(reference is again made to Example 1).

The serum albumin binders according to the different aspects of theinvention are preferably also such that they compete with the amino acidsequence of SEQ ID NO:15 for binding to (human) serum albumin and/orthat they “cross-block” (as defined herein) the binding of the aminoacid sequence of SEQ ID NO:15 to (human) serum albumin.

In particular, according to one specific aspect of the invention, theserum albumin binders according to the different aspects of theinvention are preferably such that they bind (at least) to a non-linearepitope that appears to comprise one or more of the amino acid residueswithin one or more of the following stretches of stretches of amino acidresidues within the primary sequence of human serum albumin: positions298-311 (and in particular one or more of Met298, Pro299, Ala300,Asp301, Leu302, Pro303, Ser304, Leu305, Ala306 and Glu311); positions334 to 341 (and in particular one or more of Tyr334, Arg337, His338,Pro339 and/or Asp340) and/or positions 374-381 (and in particular one ormore of Phe374, Asp375, Phe 377, Lys378 and Val381), with the amino acidresidues in human serum albumin being numbered according to thenumbering given in Meloun et al., FEBS Letters, 1975, 58, p. 134-137.Reference is given to the crystal structure data given in Example 7below.

Without being limited to any specific hypothesis or mechanism, based onthe crystal structure data given in the Experimental Part below, it isassumed that these amino acid residues on human serum albumin are partof the epitope to which the serum albumin binder of SEQ ID NO:15 bindsand that the mentioned amino acid interactions are some of the mostimportant interactions involved in this binding. Thus, preferably, thealbumin binders of the invention are such that they bind to essentiallythe same amino acid residues and/or epitope on human serum albumin asSEQ ID NO:15, and even more preferably such that they share essentiallythe same amino acid interactions SEQ ID NO:15. For this purpose,according to a specific but non-limiting aspect, the albumin binders ofthe invention preferably either have the same CDRs as the sequence ofSEQ ID NO:15, or compared to the sequence of SEQ ID NO:15 preferablycontain within their CDR's only such mutations (such as conservativeamino acid substitutions) that still allow them to undergo the same oressentially the same amino acid interactions with human serum albumin asSEQ ID NO:15.

As can also be seen from the crystal structure data given in Example 7below, some of the amino acid residues within SEQ ID NO:15 that appearto play a particularly important role in the interaction with theputative epitope on human serum albumin are those that are indicated inbold/underline in the sequence of SEQ ID NO:15 given below:

[SEQ ID NO: 15] EVQLVESGGGLVQAGGSLRLSCAASGLTFSSY AMGWFRQAPGKERERVVSIS RGGGYTYYADSV KGRFTISRDNAENTVYLQMNSLKPEDTAVYYC AAAR Y WATGS EYEFDYWGQGTLVTVSS

Some of the other amino acid residues on human serum albumin and the SEQID NO:15, respectively, that based on crystal structure data are assumedto be involved in their binding interaction, as well as some of theirassumed interactions between the amino acid residues in their respectivesequences, are given in FIG. 10 (see again also Example 7).

The serum albumin binders according to the different aspects of theinvention are generally preferably also such that they arecross-reactive between human serum albumin and serum albumin from atleast one, preferably from at least two, more preferably from at leastthree and up to essentially all of the following species of mammal: rat,mouse, rabbit, guinea pig, pig, sheep, cow and cynomolgus monkey. Inparticular, the serum albumin binders according to the different aspectsof the invention may be such that they are (at least) cross-reactivebetween human serum albumin and at least one, preferably at least two,more preferably at all three of rat serum albumin, mouse serum albuminand serum albumin from cynomolgus monkey. In this respect, the serumalbumin binders of the invention may have improved cross-reactivity (inparticular between human serum albumin on the one hand and rat and/ormouse serum albumin on the other hand) compared to serum albumin bindersthat have (essentially) the same CDR's as Alb-11 and/or Alb-23D.

For the sake of reference, FIG. 11 gives an alignment of serum albuminfrom different species of mammal (source:macromoleculeinsights.com/albumin.php, the amino acid numbering in FIG.11 is the numbering used on said webpage). For the sake of convenience,in the sequence of human serum albumin, the stretches of amino acidsthat are assumed to be part of the putative epitope of the amino acidsequences of the invention have been highlighted. Without being limitedto any specific mechanism or hypothesis, it is assumed that the aminoacid sequences of the invention are (essentially) capable of binding to(one or more amino acid residues within) the corresponding stretches ofamino acid residues that are present within the amino acid sequence ofthose mammalian serum albumins that the amino acid sequences of theinvention are cross-reactive with.

Generally, a serum albumin binder of the invention can be considered tobe cross-reactive between human serum albumin and serum albumin from oneof these species when it can bind to human serum albumin with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM; and also to the serum albumin from said species with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, again both as determined using ProteOn (reference isagain made to Example 1).

The serum albumin binders according to the different aspects of theinvention are preferably also such that either:

-   -   they have a serum half-life in man (expressed as t½ beta) that        is more than 6 hours, preferably more than 12 hours, more        preferably of more than 24 hours, even more preferably more than        72 hours; for example of about one week, two weeks and up to the        half-life of serum albumin in man (estimated to be around 19        days);        and/or such that:    -   when it is linked to a therapeutic moiety or entity, it confers        to the resulting polypeptide of the invention a serum half-life        in man (expressed as t½ beta) that is more than 6 hours,        preferably more than 12 hours, more preferably of more than 24        hours, even more preferably more than 72 hours; for example of        about one week, two weeks and up to the half-life of serum        albumin in man (estimated to be around 19 days)

The half-life in mammalian species other than man will, among otherfactors, mainly depend on the binding properties (such as affinity) ofthe albumin binder of the invention for the serum albumin from saidmammalian species as well on the half-life of the naïve serum albumin insaid species. According to a preferred embodiment of the invention, whena serum albumin binder of the invention is cross-reactive (as definedherein) between human serum albumin and serum albumin from anothermammalian species, then the half-life of the serum albumin binder of theinvention (and/or of a compound of the invention comprising said serumalbumin binder) as determined in said species is preferably at least 5%,such as at least 10%, more preferably at least 25%, for example about50% and possibly up to 100% of the half-life of the naïve serum albuminin said species.

Compared to the sequence of SEQ ID NO:15, the serum albumin binders ofthe invention preferably also contain (at least):

-   -   one or more humanizing substitutions;        and/or    -   one or more mutations (i.e. amino acid substitutions, deletions        or additions, and in particular substitutions) that reduce the        binding by pre-existing antibodies;        and may optionally contain one or more further mutations as        described herein.

For suitable humanizing substitutions (and suitable combinationsthereof), reference is for example made to WO 09/138519 (or in the priorart cited in WO 09/138519) and WO 08/020079 (or in the prior art citedin WO 08/020079), as well as Tables A-3 to A-8 from WO 08/020079 (whichare lists showing possible humanizing substitutions). Some preferred butnon-limiting examples of such humanizing substitutions are Q108L andA14P or a suitable combination thereof. Such humanizing substitutionsmay also be suitably combined with one or more other mutations asdescribed herein (such as with one or more mutations that reduce bindingby pre-existing antibodies).

For suitable mutations that can reduce the binding by pre-existingantibodies (and suitable combinations of such mutations), reference isfor example made to WO 2012/175741 and WO 2015/173325 and also to forexample WO 2013/024059 and WO 2016/118733. As described therein, suchmutations can comprise (a suitable combination of) one or more aminoacid substitutions, deletions or additions (and in particularsubstitutions), which mutations will often be in the so-calledC-terminal region of the ISV. For example, such mutations can comprisemutations (and in particular substitutions) at one or more of positions11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103,108 and/or mutations at one or more positions in the C-terminal VTVSSsequence (i.e. positions 109, 110, 111, 112 and 113), with one or moremutations at positions 11, 89, 110 and/or 112 being particularlypreferred. Some preferred but non-limiting examples of such mutationsare suitable substitutions (where required) such that after themutation, at the indicated position, one of the following amino acidresidues is present: 11L, 11K, 11V, 14A, 14P, 41A, 41L, 41P, 41S, 41T,42E, 42G, 87A, 87T, 89A, 89L, 89T, 108L, 110K, 110Q, 112K and/or 112Q(with 11L, 89A, 89L, 89T, 110K, 110Q, 112K and 112Q being particularlypreferred); or any suitable combination of such substitutions, such asfor example and without limitation: 11V in combination with 89L or 89T;11V in combination with 110K or 110Q; or 11V in combination with 89L and110K or 110Q. Such mutations that reduce binding by pre-existingantibodies may also be suitably combined with one or more othermutations as described herein (such as with one or more humanizingsubstitutions).

Where appropriate (as further described herein, and in particular whenthe serum albumin binder of the invention is present at and/or forms theC-terminal end of the compound of the invention in which it is present),for reducing the binding of pre-existing antibodies, the serum albuminbinders of the invention (and, as further described herein, also thecompounds of invention) may also comprise a C-terminal extension (suchas a C-terminal alanine residue). As described in WO 2012/175741, such aC-terminal extension reduces binding by pre-existing antibodies. Asuitable C-terminal extension can generally be further described hereinand can in particular have the formula —(X)_(n), in which X can be anynaturally occurring amino acid (but preferably not cysteine) and n canbe 1, 2, 3, 4 or 5. Reference is again made to WO 2012/175741, to alsoto for example WO 2015/173325, WO 2013/024059 and WO 2016/118733. Thepresence of such a C-terminal extension may also be suitably combinedwith one or more of the other mutations described herein (such as withone or more humanizing substitutions and/or one or more mutations thatreduce binding by pre-existing antibodies).

Other mutations that may be present in the serum albumin binders of theinvention for example and without limitation include one or moremutations (an in particular substitutions) that improve expression in adesired host cell or host organism, one or more mutations (and inparticular substitutions) that improve stability and/or resistance todegradation and/or proteases, and/or one or more other mutations thatare intended to optimize the sequence of the amino acid sequences of theinvention (for example and without limitation, one or more mutationsthat (further) reduce any tendency of the albumin binders to formdimers); or any suitable combination of such mutations.

Some non-limiting examples of such mutations are suitable substitutions(where required) such that after the mutation, at the indicatedposition, one of the following amino acid residues is present: 5V, 74S,75K, 76N and 83R; or any suitable combination of such substitutions (forexample so as to form an SKN motif at positions 75-76). Also, whereappropriate (as further described herein), the serum albumin binders ofthe invention may have a D at position 1 (i.e. a E1D mutation comparedto the sequence of SEQ ID NO:15), in particular when the serum albuminbinder of the invention is present at and/or forms the N-terminal end ofthe compound of the invention in which it is present. Such mutations mayagain be suitably combined with one or more other mutations as describedherein (such as with one or more humanizing substitutions and/or one ormore mutations that reduce binding by pre-existing antibodies).

Other mutations that may be present in the amino acid sequences of theinvention will be clear to the skilled person based on the disclosureherein.

It is also possible that a single mutation (or a suitable combination ofmutations) provides multiple functionalities or advantages. For exampleand without limitation, a humanizing Q108L substitution may also reducebinding by pre-existing antibodies.

Some preferred but non-limiting examples of amino acid residues (i.e.mutations compared to the amino acid sequence of SEQ ID NO:15) that maybe present in the amino acid sequences of the invention (i.e. bythemselves or in suitable combination) include: 11V (i.e. L11V), 14P(i.e. A14P), 47F (i.e. R47F), 49A (i.e. V49A), 74S (i.e. A74S), 75N(i.e. E75N), 83R (i.e. K83R), 89L (i.e. V89L), 89T (i.e. V89T), 110K(e.g. T110K) or 110Q (e.g. T110Q); as well as, where appropriate (asfurther described herein), 1D (e.g. E1D) and/or a C-terminal extension(X)_(n) as defined herein (such as 114A). Reference is also made tosequences and mutations shown in FIGS. 4A and 4B. For example, somepreferred but non-limiting examples of suitable combinations of suchamino acid residues (i.e. mutations compared to the amino acid sequenceof SEQ ID NO:15) include:

-   -   L11V,A14P,A74S,K83R,V89L;    -   Li1V,A14P,R47F,A74S,K83R,V89L;    -   Li1V,A14P,V49A,A74S,K83R,V89L;    -   Li1V,A14P,A74S,E75K,K83R,V89L;    -   L11V,A14P,R47F,V49A,A74S,K83R,V89L;    -   L11V,A14P,R47F,V49A,A74S,K83R,V89L;    -   L11V,A14P,V49A,A74S,E75K,K83R,V89L; or    -   L11V,A14P,R47F,V49A,A74S,E75K,K83R,V89L;        and other suitable combinations will be clear to the skilled        person based on the disclosure herein.

Some preferred, but non-limiting examples of the amino acid sequences ofthe invention are given in FIG. 2 as:

-   -   SEQ ID NOs: 15 to 35, which are examples of amino acid sequences        of the invention without a C-terminal alanine extension;    -   SEQ ID NOs: 36 to 56, which are examples of amino acid sequences        of the invention with a C-terminal extension (in each case,        exemplified by means of a C-terminal alanine extension, which is        generally the preferred C-terminal extension); and    -   SEQ ID NOs: 57 to 77, which are examples of amino acid sequences        of the invention with an N-terminal E1D mutation).

Based on the further disclosure herein, it will be clear to the skilledperson that in practice:

-   -   albumin binders of the invention with a C-terminal extension        (such as those of SEQ ID NOs: 36 to 56) will often be used        as/present at the C-terminal end of the polypeptides of the        invention (as defined herein) in which they are present;    -   albumin binders of the invention with an E1D mutation (such as        those of SEQ ID NOs: 57 to 77) will often be used as/present at        the N-terminal end of the polypeptides of the invention in which        they are present;    -   albumin binders of the invention without a C-terminal extension        and without an E1D mutation (such as those of SEQ ID NOs: 15        to 35) will often be present somewhere in the “middle” of a        polypeptide of the invention.

Each of the amino acid sequences of SEQ ID NOs: 15 to 77, as well asproteins, polypeptides and other compounds and constructs comprising thesame (as further described herein), form further aspects of the presentinvention.

In a further aspect, the invention relates to an amino acid sequencewhich is one of the amino acid sequences of SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49 or one of the amino acidsequences of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76 or SEQ ID NO:77; and each of these amino acidsequences of the invention (as well as polypeptides of the invention—asdefined herein—that comprise such an amino acid sequence of theinvention) forms a further aspect of the present invention.

As further described herein, the amino acid sequences provided by theinvention are proteins that can bind to, and that can in particularspecifically (as described herein) bind to, human serum albumin. Thus,they can be used as binding units or binding domains for binding to(human) serum albumin, for example to confer an increase in half-life(as defined herein) to therapeutic compounds, moieties or entities. Forthe use of serum albumin-binding domains to increase half-life oftherapeutic compounds, moieties or entities, reference is for examplemade to WO 2004/041865, WO 2006/122787, EP 2 139 918, WO 2011/006915, WO2012/175400 and/or WO 2014/111550. The serum albumin binders of theinvention can generally be used in the same way and for the samepurposes as the serum albumin binders described in these references.

In some further non-limiting aspects, the invention also relates to:

-   -   proteins, polypeptides and other constructs, molecules or        chemical entities that comprise or essentially consist of at        least one serum albumin binder of the invention as described        herein (again, also referred to herein as “compounds of the        invention” or as “polypeptides of the invention”);    -   methods for expressing/producing a serum albumin binder of the        invention and/or a compound of the invention;    -   a host cell, host organism or other (expression) system that can        express or produce a serum albumin binder of the invention        and/or a compound of the invention;    -   compositions and products (such as pharmaceutical compositions        and products) that comprise a serum albumin binder of the        invention and/or a compound of the invention;    -   nucleotide sequences and nucleic acids, such as (expression)        vectors, that encode a serum albumin binder of the invention        and/or a compounds of the invention;    -   uses of the compounds of the invention and/or the compounds of        the invention, such as the use of a compound of the invention to        increase the (serum) half-life of a therapeutic compounds,        moiety or entity and the therapeutic and/or prophylactic use of        a compound of the invention.

These and further aspects, embodiments, advantages, applications anduses of the invention will become clear from the further descriptionherein.

In the present specification:

-   -   the term “immunoglobulin single variable domain” (also referred        to as “ISV” or “ISVD”) is generally used to refer to        immunoglobulin variable domains (which may be heavy chain or        light chain domains, including VH, VHH or VL domains) that can        form a functional antigen binding site without interaction with        another variable domain (e.g. without a VH/VL interaction as is        required between the VH and VL domains of conventional 4-chain        monoclonal antibody). Examples of ISVDs will be clear to the        skilled person and for example include Nanobodies (including a        VHH, a humanized VHH and/or a camelized VHs such as camelized        human VHs), IgNAR, domains, (single domain) antibodies (such as        dAbs™) that are VH domains or that are derived from a VH domain        and (single domain) antibodies (such as dAbs™) that are VL        domains or that are derived from a VL domain. Unless explicitly        mentioned otherwise herein, ISVDs that are based on and/or        derived from heavy chain variable domains (such as VH or VHH        domains) are generally preferred. Most preferably, unless        explicitly indicated otherwise herein, an ISVD will be a        Nanobody.    -   the term “Nanobody” is generally as defined in WO 2008/020079 or        WO 2009/138519, and thus in a specific aspect generally denotes        a VHH, a humanized VHH or a camelized VH (such as a camelized        human VH) or generally a sequence optimized VHH (such as e.g.        optimized for chemical stability and/or solubility, maximum        overlap with known human framework regions and maximum        expression). It is noted that the terms Nanobody or Nanobodies        are registered trademarks of Ablynx N.V. and thus may also be        referred to as Nanobody® and/or Nanobodies®);    -   Generally, unless indicated otherwise herein, the ISVD's,        Nanobodies, polypeptides, proteins and other compounds and        constructs referred to herein will be intended for use in        prophylaxis or treatment of diseases or disorders in man (and/or        optionally also in warm-blooded animals and in particular        mammals). Thus, generally, the ISVD's, Nanobodies, polypeptides,        proteins and other compounds and constructs described herein are        preferably such that they can be used as, and/or can suitably be        a part of, a (biological) drug or other pharmaceutically or        therapeutically active compound and/or of a pharmaceutical        product or composition. Such a drug, compound or product is        preferably such that it is suitable for administration to a        human being, e.g. for prophylaxis or treatment of a subject in        need of such prophylaxis or treatment or for example as part of        a clinical trial. As further described herein, for this purpose,        such a drug or compound may contain other moieties, entities or        binding units besides the ISVDs provided by the invention        (which, as also described herein, may for example be one or more        other further therapeutic moieties and/or one or more other        moieties that influence the pharmacokinetic or pharmacodynamic        properties of the ISVD-based or Nanobody-based biological, such        as its half-life). Suitable examples of such further therapeutic        or other moieties will be clear to the skilled person, and for        example generally can include any therapeutically active        protein, polypeptide or other binding domain or binding unit, as        well as for example modifications such as those described on        pages 149 to 152 of WO 2009/138159. An ISVD-based biological or        Nanobody-based biological is preferably a therapeutic or        intended for use as a therapeutic (which includes prophylaxis        and diagnosis) and for this purpose preferably contains at least        one ISVD against a therapeutically relevant target (such as for        example RANK-L, vWF, IgE, RSV, CXCR4, IL-23 or other        interleukins, etc.). For some specific but non-limiting examples        of such ISVD-based or Nanobody-based biologicals, reference is        to Examples 8 to 18 and also for example made to the various        applications by Ablynx N.V. (such as for example and without        limitation WO 2004/062551, WO 2006/122825, WO 2008/020079 and WO        2009/068627), as well as for example (and without limitation) to        applications such as WO 2006/038027, WO 2006/059108, WO        2007/063308, WO 2007/063311, WO 2007/066016 and WO 2007/085814.        Also, as further described herein, the further moiety may be an        ISVD or Nanobody as described herein directed against a (human)        serum protein such as (human) serum albumin, and such an ISVD or        Nanobody may also find therapeutic uses, in particular in and/or        for extending the half-life of the TNF binders described herein.        Reference is for example made to WO 2004/041865, WO 2006/122787        and WO 2012/175400, which generally describe the use of        serum-albumin binding Nanobodies for half-life extension. Also,        in the present specification, unless explicitly mentioned        otherwise herein, all terms mentioned herein have the meaning        given in WO 2009/138519 (or in the prior art cited in WO        2009/138519) or WO 2008/020079 (or in the prior art cited in WO        2008/020079). Also, where a method or technique is not        specifically described herein, it can be performed as described        in WO 2009/138519 (or in the prior art cited in WO 2009/138519)        or WO 2008/020079 (or in the prior art cited in WO 2008/020079).        Also, as described herein, any pharmaceutical product or        composition comprising any ISVD or compound of the invention may        also comprise one or more further components known per se for        use in pharmaceutical products or compositions (i.e. depending        on the intended pharmaceutical form) and/or for example one or        more other compounds or active principles intended for        therapeutic use (i.e. to provide a combination product).

Also, when used in the present specification or claims, the followingterms have the same meaning as given on, and/or where applicable can bedetermined in the manner described in, pages 62-75 of WO 2009/138519:“agonist”, “antagonist”, “inverse agonist”, “non-polar, uncharged aminoacid residue”, “polar uncharged amino acid residue”, “polar, chargedamino acid residue”, “sequence identity”, “exactly the same” and “aminoacid difference” (when referring to a sequence comparison of two aminoacid sequences), “(in) essentially isolated (form)”, “domain”, “bindingdomain”, “antigenic determinant”, “epitope”, “against” or “directedagainst” (an antigen), “specificity” and “half-life”. In addition, theterms “modulating” and “to modulate”, “interaction site”, “specificfor”, “cross-block”, “cross-blocked” and “cross-blocking” and“essentially independent of the pH” are as defined on (and/or can bedetermined as described on) pages 74-79 of WO 2010/130832 of Ablynx N.V.Also, when referring to a construct, compound, protein or polypeptide ofthe invention, terms like “monovalent”, “bivalent” (or “multivalent”),“bispecific” (or “multispecific”), and “biparatopic” (or“multiparatopic”) may have the meaning given in WO 2009/138519, WO2010/130832 or WO 2008/020079.

The term “half-life” as used herein in relation to an ISVD, Nanobody,ISVD-based biological, Nanobody-based biological or any other amino acidsequence, compound or polypeptide referred to herein can generally bedefined as described in paragraph o) on page 57 of WO 2008/020079 and asmentioned therein refers to the time taken for the serum concentrationof the amino acid sequence, compound or polypeptide to be reduced by50%, in vivo, for example due to degradation of the sequence or compoundand/or clearance or sequestration of the sequence or compound by naturalmechanisms. The in vivo half-life of an amino acid sequence, compound orpolypeptide of the invention can be determined in any manner known perse, such as by pharmacokinetic analysis. Suitable techniques will beclear to the person skilled in the art, and may for example generally beas described in paragraph o) on page 57 of WO 2008/020079. As alsomentioned in paragraph o) on page 57 of WO 2008/020079, the half-lifecan be expressed using parameters such as the t½-alpha, t½-beta and thearea under the curve (AUC). In this respect it should be noted that theterm “half-life” as used herein in particular refers to the t½-beta orterminal half-life (in which the t½-alpha and/or the AUC or both may bekept out of considerations). Reference is for example made to theExperimental Part below, as well as to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and Peters et al, Pharmacokinetic analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).Similarly, the terms “increase in half-life” or “increased half-life”are also as defined in paragraph o) on page 57 of WO 2008/020079 and inparticular refer to an increase in the t½-beta, either with or withoutan increase in the t½-alpha and/or the AUC or both.

When a term is not specifically defined herein, it has its usual meaningin the art, which will be clear to the skilled person. Reference is forexample made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10th Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein.

Also, as already indicated herein, the amino acid residues of a Nanobodyare numbered according to the general numbering for VHs given by Kabatet al. (“Sequence of proteins of immunological interest”, US PublicHealth Services, NIH Bethesda, Md., Publication No. 91), as applied toVHH domains from Camelids in the article of Riechmann and Muyldermans,J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195; or referred toherein. According to this numbering, FR1 of a Nanobody comprises theamino acid residues at positions 1-30, CDR1 of a Nanobody comprises theamino acid residues at positions 31-35, FR2 of a Nanobody comprises theamino acids at positions 36-49, CDR2 of a Nanobody comprises the aminoacid residues at positions 50-65, FR3 of a Nanobody comprises the aminoacid residues at positions 66-94, CDR3 of a Nanobody comprises the aminoacid residues at positions 95-102, and FR4 of a Nanobody comprises theamino acid residues at positions 103-113. [In this respect, it should benoted that—as is well known in the art for VH domains and for VHHdomains—the total number of amino acid residues in each of the CDR's mayvary and may not correspond to the total number of amino acid residuesindicated by the Kabat numbering (that is, one or more positionsaccording to the Kabat numbering may not be occupied in the actualsequence, or the actual sequence may contain more amino acid residuesthan the number allowed for by the Kabat numbering). This means that,generally, the numbering according to Kabat may or may not correspond tothe actual numbering of the amino acid residues in the actual sequence.Generally, however, it can be said that, according to the numbering ofKabat and irrespective of the number of amino acid residues in theCDR's, position 1 according to the Kabat numbering corresponds to thestart of FR1 and vice versa, position 36 according to the Kabatnumbering corresponds to the start of FR2 and vice versa, position 66according to the Kabat numbering corresponds to the start of FR3 andvice versa, and position 103 according to the Kabat numberingcorresponds to the start of FR4 and vice versa.].

Alternative methods for numbering the amino acid residues of VH domains,which methods can also be applied in an analogous manner to VHH domainsfrom Camelids and to Nanobodies, are the method described by Chothia etal. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and theso-called “contact definition”. However, in the present description,aspects and figures, the numbering according to Kabat as applied to VHHdomains by Riechmann and Muyldermans will be followed, unless indicatedotherwise.

It should also be noted that the Figures, any Sequence Listing and theExperimental Part/Examples are only given to further illustrate theinvention and should not be interpreted or construed as limiting thescope of the invention and/or of the appended claims in any way, unlessexplicitly indicated otherwise herein.

As further described herein, the serum albumin binders of the inventioncan be used with advantage as a moiety, binding unit or fusion partnerin order to increase the half-life of therapeutic compounds, moieties orentities such as polypeptides, proteins, compounds (including, withoutlimitation, small molecules) or other therapeutic entities.

Thus, in another aspect, the invention provides polypeptides, proteins,constructs, compounds or other chemical entities that comprise oressentially consist of a serum albumin binder of the invention and oneor more other amino acid sequences, (binding) domains, binding units orother moieties or chemical entities.

In particular, the invention provides polypeptides, proteins,constructs, compounds or other chemical entities that comprise a serumalbumin binder of the invention and one or more (such as one or two)therapeutic moieties (which may be the same or different, and may forexample be directed against the same target or to different targets, andwhen they are directed to the same target may be directed towards thesame or different epitopes, parts, domains or subunits of said target),suitably linked to each other either directly or via one or moresuitable linkers or spacers. Such polypeptides, proteins or constructsmay for example and without limitation be a fusion protein, as furtherdescribed herein.

The invention further relates to therapeutic uses of such polypeptides,proteins, constructs or compounds and to pharmaceutical compositionscomprising such polypeptides, proteins, constructs or compounds.

In one aspect, the at least one therapeutic moiety comprises oressentially consists of a therapeutic protein, polypeptide, compound,factor or other entity. In a preferred embodiment the therapeutic moietyis directed against a desired antigen or target, is capable of bindingto a desired antigen (and in particular capable of specifically bindingto a desired antigen), and/or is capable of interacting with a desiredtarget. In another embodiment, the at least one therapeutic moietycomprises or essentially consists of a therapeutic protein orpolypeptide. In a further embodiment, the at least one therapeuticmoiety comprises or essentially consists of a binding domain or bindingunit, such as an immunoglobulin or immunoglobulin sequence (includingbut not limited to a fragment of an immunoglobulin), such as an antibodyor an antibody fragment (including but not limited to an ScFv fragment),or of another suitable protein scaffold, such as protein A domains (suchas Affibodies™) tendamistat, fibronectin, lipocalin, CTLA-4, T-cellreceptors, designed ankyrin repeats, avimers and PDZ domains (Binz etal., Nat. Biotech 2005, Vol 23:1257), and binding moieties based on DNAor RNA including but not limited to DNA or RNA aptamers (Ulrich et al.,Comb Chem High Throughput Screen 2006 9(8):619-32).

In yet another aspect, the at least one therapeutic moiety comprises oressentially consists of an antibody variable domain, such as a heavychain variable domain or a light chain variable domain.

In a preferred aspect, the at least one therapeutic moiety comprises oressentially consists of at least one immunoglobulin single variabledomain, such as a domain antibody, single domain antibody, “dAb” orNanobody (such as a VHH, a humanized VHH or a camelized VH) or an IgNARdomain.

In a specific embodiment, the at least one therapeutic moiety comprisesor essentially consists of at least one monovalent Nanobody or abivalent, multivalent, bispecific or multispecific Nanobody construct.

The polypeptides, (fusion) proteins, constructs or compounds thatcomprise a serum albumin binder of the invention and one or moretherapeutic moieties can generally be (prepared and used) as describedin the prior art cited above (such as WO 04/041865, WO 06/122787, WO2012/175400 and WO 2015/173325; reference is also made to for example,WO 2004/003019, EP 2 139 918, WO 2011/006915 and WO 2014/111550) with aserum albumin binder of the invention instead of the half-lifeincreasing moieties described in said

The polypeptides, (fusion) proteins, constructs or compounds thatcomprise a serum albumin binder of the invention and one or moretherapeutic moieties will generally and preferably have an increasedhalf-life (as described herein, and preferably expressed as t½-beta),compared to the therapeutic moiety or moieties per se.

Generally, the compounds, polypeptides, constructs or fusion proteinsdescribed herein preferably have a half-life (again, as describedherein, and preferably expressed as t½-beta) that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding therapeutic moiety per se (as measured in either in man ora suitable animal, such as mouse or cynomolgus monkey).

Also, preferably, any such compound, polypeptide, fusion protein orconstruct has a half-life (again, as described herein, and preferablyexpressed as t½-beta) in man that is increased with more than 1 hour,preferably more than 2 hours, more preferably of more than 6 hours, suchas of more than 12 hours, compared to the half-life of the correspondingtherapeutic moiety per se.

Also, preferably, a compound or polypeptide of the invention has ahalf-life (again, as described herein, and preferably expressed ast½-beta) in man that is more than 1 hour, preferably more than 2 hours,more preferably of more than 6 hours, such as of more than 12 hours, andfor example of about one day, two days, one week, two weeks and up tothe half-life of serum albumin in man (estimated to be around 19 days).

As mentioned, in one aspect, a serum albumin binder of the invention isused to increase the half-life of (one or more) immunoglobulin singlevariable domains, such as domain antibodies, single domain antibodies,“dAb's”, VHH's or Nanobodies (such as VHH's, humanized VHH's orcamelized VH's such as camelized human VH's).

Thus, one embodiment of the invention relates to a polypeptide,construct or fusion protein that comprises a serum albumin binder of theinvention and one or more (such as one or two) immunoglobulin singlevariable domain sequences, which are suitably linked to each other,either directly or optionally via one or more suitable linkers orspacers. As mentioned herein, each such immunoglobulin single variabledomain present in such a polypeptide, construct or fusion protein mayindependently be a domain antibody, single domain antibody, “dAb′” orNanobody (such as a VHH, humanized VHH or camelized VH, such as acamelized human VH); and according to one specific but non-limitingaspect, at least one (and up to all) of these immunoglobulin singlevariable domains comprises two or three disulphide bridges. Preferably,all ISVDs present in such a compound of the invention are Nanobodies.

When a compound of the invention has an ISVD at its C-terminal end (suchas a serum albumin binder of the invention or an ISVD that is directedagainst a therapeutic target), then said C-terminal ISVD (and thus, byextension, the entire compound of the invention) preferably has aC-terminal extension at its C-terminal end. This C-terminal extensionwill be directly linked to the last C-terminal amino acid residue of theISVD, which will usually be the amino acid residue at position 113according to Kabat (unless the ISVD contains one or more amino aciddeletions such that the sequence of the ISVD ends before position 113).Thus, generally, the C-terminal extension will be directly linked to theC-terminal VTVSS sequence (SEQ ID NO:78) of the C-terminal ISV (andthus, by extension, to the C-terminal TVTSS sequence of the compound ofthe invention) or the C-terminal sequence of the C-terminal ISVD thatcorresponds to the C-terminal ISVD sequence (for example, where saidC-terminal sequence of the C-terminal ISVD contains one or moresubstitutions or deletions compared to the usual VTVSS sequence, such asT110K, T110Q, S112K or S112K).

It will also be clear to the skilled person in the case where a compoundof the invention has a serum albumin binder of the invention at itsC-terminal end, that then said serum albumin binder of the inventionwill carry said C-terminal extension.

Generally, any C-terminal extension that is used herein (i.e. at theC-terminal end of a compound of the invention and/or at the C-terminalend of a serum albumin binder of the invention) can generally be asdescribed in WO 2012/174741 or WO 2015/173325 (reference is also made tofor example WO 2103/024059 and WO2016/118733). In particular, aC-terminal extension may have the formula (X)_(n), in which n is 1 to10, preferably 1 to 5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2,such as 1); and each X is an (preferably naturally occurring) amino acidresidue that is independently chosen from naturally occurring amino acidresidues (although according to preferred one aspect, it does notcomprise any cysteine residues), and preferably independently chosenfrom the group consisting of alanine (A), glycine (G), valine (V),leucine (L) or isoleucine (I).

According to some preferred, but non-limiting aspects of such C-terminalextensions X_((n)), X and n can be as follows:

-   (a) n=1 and X=Ala;-   (b) n=2 and each X=Ala;-   (c) n=3 and each X=Ala;-   (d) n=2 and at least one X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (e) n=3 and at least one X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (f) n=3 and at least two X=Ala (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (g) n=1 and X=Gly;-   (h) n=2 and each X=Gly;-   (i) n=3 and each X=Gly;-   (j) n=2 and at least one X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (k) n=3 and at least one X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (l) n=3 and at least two X=Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);-   (m) n=2 and each X=Ala or Gly;-   (n) n=3 and each X=Ala or Gly;-   (o) n=3 and at least one X=Ala or Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile); or-   (p) n=3 and at least two X=Ala or Gly (with the remaining amino acid    residue(s) X being independently chosen from any naturally occurring    amino acid but preferably being independently chosen from Val, Leu    and/or Ile);    with aspects (a), (b), (c), (g), (h), (i), (m) and (n) being    particularly preferred, with aspects in which n=1 or 2 being    preferred and aspects in which n=1 being particularly preferred.

It should also be noted that, preferably, any C-terminal extensionpresent in a serum albumin binder of the invention does not contain a(free) cysteine residue (unless said cysteine residue is used orintended for further functionalization, for example for pegylation).

Some specific, but non-limiting examples of useful C-terminal extensionsare the following amino acid sequences: A, AA, AAA, G, GG, GGG, AG, GA,AAG, AGG, AGA, GGA, GAA or GAG.

Preferably also, when a compound of the invention has an ISVD at itsC-terminal end (such as a serum albumin binder of the invention or anISVD that is directed against a therapeutic target), then (at least)said C-terminal ISVD preferably contains, even more preferably inaddition to a C-terminal extension as described herein, one or moremutations that reduce binding by pre-existing antibodies (i.e. asdescribed herein for the serum albumin binders of the invention and asmore generally described in WO 2012/175741 and WO 2015/173325 and alsofor example in WO 2013/024059 and WO 2016/118733). In this respect, itwill be clear to the skilled person in the case where a compound of theinvention has a serum albumin binder of the invention at its C-terminalend, that then (at least) said serum albumin binder of the inventionpreferably will contain such mutations (i.e. preferably in addition to aC-terminal extension).

More generally, according to a specific aspect of the invention, when acompound of the invention contains two or more ISVDs (e.g. a serumalbumin binder of the invention and one or more ISVDs against atherapeutic target), then preferably all these ISVDs contain mutationsthat reduce binding to pre-existing antibodies (again, preferably inaddition to the C-terminal extension that is linked to the C-terminalISVD if the compound of the invention has an ISVD at its C-terminalend).

When a compound of the invention has an ISVD at its N-terminal end (suchas a serum albumin binder of the invention or an ISVD that is directedagainst a therapeutic target), then said N-terminal ISVD (and thus, byextension, the entire compound of the invention) preferably contain a Dat position 1. In this respect, it will again be clear to the skilledperson in the case where a compound of the invention has a serum albuminbinder of the invention at its N-terminal end, that then said serumalbumin binder of the invention will preferably have a D at position 1(e.g. an E1D mutation compared to for example the sequence of SEQ IDNO:15, such as the in the amino acid sequences of the invention of SEQID NOs: 57 to 77).

In some further aspects, the invention relates to a protein, polypeptideor other compound or construct that comprises or essentially consists ofat least one (and preferably only one) serum albumin binder of theinvention and at least one (such as one, two or three) therapeuticmoiety or entity (in which said serum albumin binder and the one or moretherapeutic moieties or entities are suitably linked, optionally via oneor more suitable linkers), which protein, polypeptide, compound,construct is such that:

-   -   when it has an ISVD at its C-terminal end, then (the C-terminal        ISVD of) said protein, polypeptide, compound, construct has a        C-terminal extension (X)_(n) (as further described herein) at        its C-terminal end; and/or    -   when it has an ISVD at its C-terminal end, then at least said        the C-terminal ISVD contains one or more mutations that reduce        the binding of pre-existing antibodies (as further described        herein);    -   when it has an ISVD at its N-terminal end, then (the N-terminal        ISVD of) said protein, polypeptide, compound, construct        preferably contains a D at position 1; and/or    -   in which said ISVDs which protein, polypeptide or other compound        may also have ISVD at its N-terminal end, in which case said        N-terminal ISVD end preferably has a D or an E1D at position 1;    -   preferably, essentially all of the ISVDs present in said        protein, polypeptide, compound, construct contain one or more        mutations that reduce the binding of pre-existing antibodies (as        further described herein).

According to one specific aspect of the invention, all therapeuticmoieties present in a compound of the invention are ISVD's (i.e. ISVDsagainst a therapeutic target), and in particular heavy-chain ISVDs, andmore in particular Nanobodies (i.e. Nanobodies against a therapeutictarget).

For example and without limitation, such compounds of the invention maycomprise:

-   -   one copy of a serum albumin binder of the invention and one ISVD        (and preferably Nanobody) against a therapeutic target; or    -   one copy of a serum albumin binder of the invention and two        ISVDs (and preferably two Nanobodies) against a therapeutic        target (which ISVDs may be the same or different and when        different may be directed against the same target, against        different epitopes on the same target or against different        therapeutic targets); or    -   one copy of a serum albumin binder of the invention and three        ISVDs (and preferably three Nanobodies) against a therapeutic        target (which ISVDs may be the same or different and when        different may be directed against the same target, against        different epitopes on the same target or against different        therapeutic targets).

Some non-limiting examples of constructs, fusion proteins orpolypeptides of the invention can be schematically represented asfollows, in which “[Alb]” represents a serum albumin binder of theinvention, “[therapeutic moiety 1]” and “[therapeutic moiety 2]”represent the therapeutic moieties (which as mentioned may eachindependently be an immunoglobulin single variable domain), “-”represents a suitable linker (which is optional; suitable examples are9GS and 35GS linkers) and the N-terminus is on the left hand side andthe C-terminus is on the right hand side:

-   -   [Alb]-[therapeutic moiety 1]    -   [therapeutic moiety 1]-[Alb]-X_((n))    -   [Alb]-[therapeutic moiety 1]-[therapeutic moiety 1]    -   [therapeutic moiety 1]-[therapeutic moiety 1]-[Alb]-X_((n))    -   [therapeutic moiety 1]-[Alb]-[therapeutic moiety 1]    -   [Alb]-[therapeutic moiety 1]-[therapeutic moiety 2]    -   [therapeutic moiety 1]-[therapeutic moiety 2]-[Alb]-X_((n))    -   [therapeutic moiety 1]-[Alb]-[therapeutic moiety 2]

When the therapeutic moieties are ISVDs (and preferably Nanobodies)against a therapeutic target, preferred but non-limiting constructs,fusion proteins or polypeptides of the invention can be schematicallyrepresented as follows, in which “[Alb]” represents a serum albuminbinder of the invention, “[therapeutic ISVD 1]” and “[therapeutic ISVD2]” represent ISVDs against a therapeutic target (which ISVDs may be thesame or different and when different may be directed against the sametarget, against different epitopes on the same target or againstdifferent therapeutic targets), “-” represents a suitable linker (whichis optional), X_((n)) represents a C-terminal extension as describedherein, and the N-terminus is on the left hand side and the C-terminusis on the right hand side:

-   -   [Alb]-[therapeutic ISVD 1]-X_((n))    -   [therapeutic ISVD 1]-[Alb]-X_((n))    -   [Alb]-[therapeutic ISVD 1]-[therapeutic ISVD 1]-X_((n))    -   [therapeutic ISVD 1]-[therapeutic ISVD 1]-[Alb]-X_((n))    -   [therapeutic ISVD 1]-[Alb]-[therapeutic ISVD 1]-X_((n))    -   [Alb]-[therapeutic ISVD 1]-[therapeutic ISVD 2]-X_((n))    -   [therapeutic ISVD 1]-[therapeutic ISVD 2]-[Alb]-X_((n))    -   [therapeutic ISVD 1]-[Alb]-[therapeutic ISVD 2]-X_((n))

Thus, in another aspect, the invention relates to a multispecific (andin particular bispecific) Nanobody construct that comprises a serumalbumin binder of the invention and at least one other Nanobody (such asone or two other Nanobodies, which may be the same or different), inwhich said at least one other Nanobody is preferably directed against adesired target (which is preferably a therapeutic target) and/or anotherNanobody that useful or suitable for therapeutic, prophylactic and/ordiagnostic purposes. Again, the serum albumin binder of the inventionand the other Nanobodies may be suitably linked to each other eitherdirectly or optionally via one or more suitable linkers or spacers.

For a general description of multivalent and multispecific polypeptidescontaining one or more Nanobodies and their preparation, reference isalso made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350,2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001),277-302; as well as to for example WO 96/34103, WO 99/23221, WO04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO2009/068627.

By means of illustration, some examples of compounds of the inventionare given in SEQ ID NOs:82 to 88, using the anti-HER2-Nanobody of SEQ IDNO: 81 as a representative example of an anti-target Nanobody, and withthe constituent Nanobodies being in different positions in the compoundof the invention. The compounds of SEQ ID NOs: 82 to 85 are examplesillustrating bivalent bispecific compounds of the invention and thecompounds of SEQ ID NOs: 86 to 88 are examples illustrating trivalentbispecific compounds of the invention. In each case, the compoundscontain an E1D mutation and a C-terminal alanine residue, and containrepresentative but non-limiting examples of the use of suitable linkers(i.e. a 15GS linker in SEQ ID NO:83 and or 35GS linkers in SEQ ID NOs:84 and 85-88).

Some other examples of some specific multispecific and/or multivalentpolypeptide of the invention can be found in the applications by AblynxN.V. mentioned herein. In particular, for a general description ofmultivalent and multispecific constructs comprising at least oneNanobody against a serum protein for increasing the half-life, ofnucleic acids encoding the same, of compositions comprising the same, ofthe preparation of the aforementioned, and of uses of theaforementioned, reference is made to the International applications WO04/041865 and WO 06/122787 mentioned above (the serum albumin binders ofthe invention described herein can generally be used analogously to thehalf-life extending Nanobodies described therein such as Alb-8), as wellas to the general description and specific examples of such constructsgiven in for example WO 04/041862, WO 2006/122786, WO 2008/020079, WO2008/142164 or WO 2009/068627.

The invention also relates to nucleotide sequences or nucleic acids thatencode the albumin binders, compounds or polypeptides of the invention.The invention further includes genetic constructs that include theforegoing nucleotide sequences or nucleic acids and one or more elementsfor genetic constructs known per se. The genetic construct may be in theform of a plasmid or vector. Again, such constructs can be generally asdescribed in the published patent applications of Ablynx N.V., such asfor example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164or WO 2009/068627.

The invention also relates to hosts or host cells that contain suchnucleotide sequences or nucleic acids, and/or that express (or arecapable of expressing), the albumin binders, compounds or polypeptidesof the invention. Again, such host cells can be generally as describedin the published patent applications of Ablynx N.V., such as for exampleWO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO2009/068627.

The invention also relates to a method for preparing an albumin binder,compound or polypeptide of the invention, which method comprisescultivating or maintaining a host cell as described herein underconditions such that said host cell produces or expresses an albuminbinder, compound or polypeptide of the invention, and optionally furthercomprises isolating the albumin binder, compound or polypeptide of theinvention so produced. Again, such methods can be performed as generallydescribed in the published patent applications of Ablynx N.V., such asfor example WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164or WO 2009/068627.

The invention also relates to a pharmaceutical composition thatcomprises at least one compound or polypeptide of the invention, andoptionally at least one pharmaceutically acceptable carrier, diluent orexcipient. Such preparations, carriers, excipients and diluents maygenerally be as described in the published patent applications of AblynxN.V., such as for example WO 04/041862, WO 2006/122786, WO 2008/020079,WO 2008/142164 or WO 2009/068627.

However, since the compounds or polypeptides of the invention have anincreased half-life, they are preferably administered to thecirculation. As such, they can be administered in any suitable mannerthat allows the compound or polypeptide of the invention to enter thecirculation, such as intravenously, via injection or infusion, or in anyother suitable manner (including oral administration, subcutaneousadministration, intramuscular administration, administration through theskin, intranasal administration, administration via the lungs, etc.).Suitable methods and routes of administration will be clear to theskilled person, again for example also from the teaching of thepublished patent applications of Ablynx N.V., such as for example WO04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164 or WO2009/068627.

Thus, in another aspect, the invention relates to a method for theprevention and/or treatment of at least one disease or disorder that canbe prevented or treated by the use of a compound or polypeptide of theinvention, which method comprises administering, to a subject in needthereof, a pharmaceutically active amount of a compound or polypeptideof the invention, and/or of a pharmaceutical composition comprising thesame. The diseases and disorders that can be prevented or treated by theuse of a compound or polypeptide of the invention as described hereinwill generally be the same as the diseases and disorders that can beprevented or treated by the use of the therapeutic moiety or moietiesthat is/are present in the compound or polypeptide of the invention.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein.

In another embodiment, the invention relates to a method forimmunotherapy, and in particular for passive immunotherapy, which methodcomprises administering, to a subject suffering from or at risk of thediseases and disorders mentioned herein, a pharmaceutically activeamount of a compound or polypeptide of the invention, and/or of apharmaceutical composition comprising the same.

The compound or polypeptide of the invention and/or the compositionscomprising the same are administered according to a regime of treatmentthat is suitable for preventing and/or treating the disease or disorderto be prevented or treated. The clinician will generally be able todetermine a suitable treatment regimen, depending on factors such as thedisease or disorder to be prevented or treated, the severity of thedisease to be treated and/or the severity of the symptoms thereof, thespecific polypeptide of the invention to be used, the specific route ofadministration and pharmaceutical formulation or composition to be used,the age, gender, weight, diet, general condition of the patient, andsimilar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more compounds or polypeptides of the invention, or of one or morecompositions comprising the same, in one or more pharmaceuticallyeffective amounts or doses. The specific amount(s) or doses to beadministered can be determined by the clinician, again based on thefactors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency and/or the half-life of thecompounds or polypeptides of the invention to be used, the specificroute of administration and the specific pharmaceutical formulation orcomposition used, the compounds or polypeptides of the invention willgenerally be administered in an amount between 1 gram and 0.01 microgramper kg body weight per day, preferably between 0.1 gram and 0.1microgram per kg body weight per day, such as about 1, 10, 100 or 1000microgram per kg body weight per day, either continuously (e.g., byinfusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

Also, as the compounds of the invention contain a half-life extendingserum albumin binder of the invention, they do not need to beadministered essentially continuously (e.g. by infusion), but they canbe administered at suitable intervals (to be determined by the skilledperson). For example, they can be administered (at a suitable dose) onceevery two days, once every four days, once weekly, once every two weeksand in some cases once every four weeks or even less frequently, forexample by injection or infusion.

One aspect of the invention relates to a pharmaceutical compositioncomprising at least one compound or polypeptide of the invention whereinsaid composition is intended for administration at an interval betweenonce weekly and once every 4 weeks, and in particular between once every7 days and once every 21 days, such as once every 7 days or 14 days.

Usually, in the above method, a single polypeptide of the invention willbe used. It is however within the scope of the invention to use two ormore polypeptides of the invention in combination.

The polypeptides of the invention may also be used in combination withone or more further pharmaceutically active compounds or principles,i.e., as a combined treatment regimen, which may or may not lead to asynergistic effect. Again, the clinician will be able to select suchfurther compounds or principles, as well as a suitable combinedtreatment regimen, based on the factors cited above and his expertjudgement.

In particular, the polypeptides of the invention may be used incombination with other pharmaceutically active compounds or principlesthat are or can be used for the prevention and/or treatment of thediseases and disorders that can be prevented or treated with the fusionproteins or constructs of the invention, and as a result of which asynergistic effect may or may not be obtained.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and or a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures, in which:

FIG. 1 is a table listing some of the amino acid positions that will bespecifically referred to herein and their numbering according to somealternative numbering systems (such as Aho and IMGT);

FIG. 2 lists the amino acid sequences referred to herein;

FIGS. 3A and 3B show an alignment of the sequence of SEQ ID NO:15(invention) with the prior art sequences of SEQ ID NOs: 1 and 2 and thereference sequences of SEQ ID NO: 79 and 80 (which are based on SEQ IDNO:1 and SEQ ID NO:2, respectively);

FIG. 4A shows an alignment of SEQ ID NOs: 15 to 56 and FIG. 4B shows analignment of SEQ ID NOs: 15 and 57 to 77;

FIG. 5 is a graph showing competitive binding for the serum albuminbinder of SEQ ID NO:1 (reference), the serum albumin binder of SEQ IDNO:15 (invention) and an irrelevant Nanobody (cAblys3-Flag3His6), asgenerated in Example 2;

FIG. 6 is a graph showing binding of human serum albumin to FcRn in thepresence of the serum albumin binder of SEQ ID NO:15. Human FcRn-human32 microglobulin heterodimer was immobilized on CM5 chip. Binding of 1μM HSA in absence or presence of 2 μM Nanobody in 50 mM NaPO4+150 mMNaCl+0.05% Tween-20 pH 6.0 was monitored on a Biacore T100 instrument;

FIG. 7 shows the data collection and processing statistics used inExample 7 in determining/calculating the crystal structure for thecrystal structure of human serum albumin and the amino acid sequence ofSEQ ID NO:15;

FIG. 8 lists the refinement statistics used for used in Example 7 indetermining/calculating the crystal structure for the crystal structureof human serum albumin and the amino acid sequence of SEQ ID NO:15;

FIG. 9 is a graph showing serum concentrations for the constructs of SEQID NO:82 (invention), SEQ ID NO:89 (reference) and SEQ ID NO:90(reference), respectively, as determined in Example 5. The symbols inthe graph represent individual data points and the lines represent themean concentration.

FIG. 10 shows the most important residues on human serum albumin and onSEQ ID NO: 15, respectively, that based on the crystal structure datagenerated in Example 7 are assumed to be involved in the bindinginteraction between human serum albumin and SEQ ID NO: 15. FIG. 10 alsoshows the main binding interactions between these respective amino acidresidues.

FIG. 11 gives an alignment of serum albumin from different species ofmammal. In the sequence of human serum albumin, the stretches of aminoacids that are assumed to be part of the putative epitope of SEQ IDNO:15 (see also FIG. 10 ) have been highlighted. Mouse: SEQ ID NO: 92;Rat: SEQ ID NO: 93; Dog: SEQ ID NO: 96; Cat: SEQ ID NO: 97; Human SEQ IDNO: 91; Cow: SEQ ID NO: 94; Sheep: SEQ ID NO: 98; Pig: SEQ ID NO: 95;Horse: SEQ ID NO: 99; Rabbit: SEQ ID NO: 100.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, and copending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

EXPERIMENTAL PART Example 1: Affinity for Serum Albumin

The affinity of the serum albumin binder of SEQ ID NO:15 for human(Sigma-Aldrich A3782), cynomolgus monkey (generated in-house), mouse(Albumin Bioscience 2601), rat (Sigma-Aldrich A4538), rabbit(Sigma-Aldrich A0764), guinea pig (Gentaur GPSA62), pig (Sigma-AldrichA4414), sheep (Sigma-Aldrich A3264) and bovine (Sigma-Aldrich A3059)serum albumin (SA) was measured via Surface Plasmon Resonance (SPR) on aProteOn XPR36 (BioRad) instrument. Serum albumin was immobilized viaamine coupling on GLC ProteOn chip using ProteOn Amine Coupling Kit(BioRad). Different concentrations (300 nM, 100 nM, 33.3 nM, 11.1 nM,3.7 nM and 1.23 nM) of the serum albumin binder of SEQ ID NO:15 wereinjected in HBS-P+ pH 7.4 buffer (GE Healthcare) at 45 μL/min for 120 s,followed by dissociation for 900 s. There was no or very low bindingobserved for the serum albumin binder of SEQ ID NO:15 on rabbit, pig,sheep and bovine SA. The affinity of the serum albumin binder of SEQ IDNO:15 for human, cynomolgus monkey, rat, mouse and guinea pig SA washigher compared to the respective affinities of serum albumin binder ofSEQ ID NO:1 (reference) as determined in a separate experiment. Theresults are shown in Table 1.

TABLE 1 Kinetic parameters for binding of the serum albumin binder ofSEQ ID NO: 15 on SA from different species. SEQ ID NO: 15 SEQ ID NO: 1(reference) SA ka (s⁻¹M⁻¹) kd (s⁻¹) KD (M) ka (s⁻¹M⁻¹) Kd (s⁻¹) KD (M)human 8.1E+05 1.5E−04 1.9E−10 4.9E+05 1.6E−03 3.3E−09 cyno 7.7E+059.4E−05 1.2E−10 4.6E+05 1.4E−03 3.1E−09 rat 1.0E+06 2.2E−02 2.1E−083.9E+05 2.6E−01 6.7E−07 mouse 1.2E+06 2.4E−03 1.9E−09 6.6E+05 3.0E−023.9E−08 guinea 1.4E+06 2.1E−03 1.5E−09 9.4E+05 1.9E−02 2.0E−08 pig

The long half-life of albumin in blood is mainly driven by twocharacteristics: (i) the large size (65 kDa) of albumin limits itsglomerular filtration and (ii) albumin binds to FcRn at low pH (pH 6),which protects albumin from degradation in the lysosomes after passiveendocytosis in endothelial and epithelial cells, by recycling from earlyendosome back to the extracellular environment. For albumin-bindingNanobodies to result in long serum half-life through albumin binding andsubsequent recycling, these should stay bound to albumin in the pH rangefrom 5.0 to 7.4. The dissociation rate of the serum albumin binder ofSEQ ID NO:15 from HSA at pH 5, pH 6 and pH 7.4 was measured on a ProteOninstrument as described above, including serum albumin binder of SEQ IDNO:1 as a reference. The serum albumin binder of SEQ ID NO:15 and serumalbumin binder of SEQ ID NO:1 (reference) were injected at 500 nM and300 nM respectively in HBS-P+ pH 7.4 buffer. Dissociation buffers were50 mM NaOAc/HOAc+150 mM NaCl+0.05% Tween-20 pH 5.0, 50 mM NaOAc/HOAc+150mM NaCl+0.05% Tween-20 pH 6.0 and HBS-P+ pH 7.4 respectively.Dissociation was analysed for 2700 s. As can be seen from the data shownin Table 2, the dissociation rates for the serum albumin binder of SEQID NO:15 do not differ significantly across the pH range from 5.0 to7.4.

TABLE 2 Dissociation rate of T023500010 from HSA at different pH. kd(s⁻¹) SEQ ID NO: 15 SEQ ID NO: 1 (reference) pH 7.4 1.1E−04 1.3E−03 pH6.0 6.9E−05 9.2E−04 pH 5.0 5.8E−05 1.1E−03 SEQ ID NO: 15 or SEQ ID NO: 1were injected on immobilized human serum albumin. Dissociation wasmonitored at pH 5.0, 6.0 and 7.4 on a ProteOn instrument.

Example 2: Epitope

Epitope binning was analysed in a competition ELISA. Human serum albuminwas coated at 125 ng/ml in PBS at 4° C. over night. After blocking withPBS+1% casein, 1.5 nM serum albumin binder of [SEQ ID NO:1-cMycHis6] anda concentration series of competitors ([His6Flag3-SEQ ID NO:15],[His6Flag3-SEQ ID NO:1] as positive control or hen egg lysozyme bindingsingle domain antibody cAblys3-Flag3His6 as negative control) wereadded. Bound [SEQ ID NO:1-cMycHis6] was detected with goat anti-cMyc(Abcam abl9234) and HRP-labelled rabbit anti-goat (Genway 18-511-244226)antibodies.

The results are shown in FIG. 5 . It was found that the serum albuminbinder of SEQ ID NO:15 and serum albumin binder of SEQ ID NO:1(reference) do not bind identical epitopes on human serum albumin.

Example 3: Interference with Interaction Between SA and FcRn

For the serum albumin binder of SEQ ID NO:15 to result in long half-lifevia albumin binding and subsequent recycling, it should not interferewith the binding of albumin to FcRn. This was analysed in SPR on aBiacore T100 (GE Healthcare) instrument. Human FcRn-human 32microglobulin heterodimer (Sino Biological CT009-H08H) was immobilizedon CM5 chip via standard amine coupling (Biacore amine coupling kit). Amixture of 1 μM HSA and 2 μM Nanobody ([His6Flag3-SEQ ID NO:15],[His6Flag3-SEQ ID NO:1](reference) or cAblys3-Flag3His6) in 50 mMNaPO4+150 mM NaCl+0.05% Tween-20 pH 6.0 was injected at 10 μl/min for120 s, followed by dissociation for 600 s. Binding curves werequalitatively compared with binding curve of 1 μM HSA in absence ofNanobody. As can be seen from FIG. 6 , the serum albumin binder of SEQID NO:15 did not interfere with the binding of HSA to FcRn.

Example 4: Physical Stability

The stability of the serum albumin binder of SEQ ID NO:15 was assessed,using the serum albumin binders of SEQ ID NOs:1 and 2 as reference.Melting temperature (Tm) was determined in Differential ScanningCalorimetry (DSC). In addition, the physical stability was analysed bymeasuring the following parameters before and after storage at 40° C. inD-PBS at 5 mg/ml: Turbidity (OD_(500nm)), percentage high molecularweight variants (SE-HPLC), content (OD280) and chemical variants(RP-HPLC).

The results are shown in Table 3. For all constructs, storage at 40° C.resulted in an increase in pre-peak in SE-HPLC (high molecular weightvariants), which was clearly lower for the serum albumin binder of SEQID NO:15 and that of SEQ ID NO:2 compared to serum albumin binder of SEQID NO:1.

TABLE 3 Summary data physical stability the serum albumin binder of SEQID NO: 15. Stability 40° C. 2 weeks Δ pre-peak Turbidity at Protein TmSE-HPLC (%) T = 2 w (OD_(500 nm)) loss (° C.) SEQ ID NO: 15 0.3 0.01 no72.4 SEQ ID NO: 1 11.8 0.1 no 66.0 (reference) SEQ ID NO: 2 0.6 0.01 no72.0 (reference) Tm was measured in DSC. The serum albumin binders werestored for 2 weeks at 40° C. at a concentration of 5 mg/mL in PBS.Turbidity, SE-HPLC profile and protein content (RP-UHPLC) were measuredbefore and after storage.

Example 5: PK Profile of the Serum Albumin Binder of SEQ ID NO:15 in Rat

The pharmacokinetics of a representative compound of the invention (SEQID NO:82) comprising the serum albumin binder of SEQ ID NO:15 aftersingle i.v. dose were studied in Sprague Dawley rats and compared tosimilar constructs (SEQ ID NOs: 89 and 90) comprising the referenceserum albumin binders of SEQ ID NOs: 79 and 80, respectively. Alignmentsof SEQ ID NO:15 with the reference sequences of SEQ ID NOs: 1, 2, 79 and80 are given in FIGS. 3A and 3B. The constructs of SEQ ID NOs: 82, 89and 90 comprise the relevant serum albumin binder linked via a 35GSlinker to a representative Nanobody (the anti-HER2 Nanobody of SEQ IDNO:81) as well as an E1D mutation and a C-terminal alanine, and wereproduced in Pichia pastoris.

Radio-iodination of the bivalent Nanobodies was conducted usingN-succinimidyl 3-¹²⁵I-iodobenzoate (¹²⁵I-SIB) in borate buffer 0.2 M pH8.3. There was no indication that labelling of the Nanobodies interferedwith binding to HSA. Rats were dosed with 20 μg of the ¹²⁵I-labelledconstruct (3 rats per group) at a specific activity of 3.5-4 mCi/mgNanobody construct. Blood samples were taken at 5 min, 1 h, 4 h, 8 h, 24h, 48 h, 96 h, 168 h, 240 h, 336 h and 504 h post dosing. Radioactivitywas measured in each blood sample and converted to a proteinconcentration based on the specific activity of the labelled Nanobodyconstruct. The decay of the radioactive label over time was taken intoaccount in the calculations. The measured concentrations of theconstructs in blood over time are shown in FIG. 9 . PK parameters werecalculated by non-compartmental analysis: the relevant data are listedin Table 4). In line with the expected higher affinity for rat SAcompared to the references of SEQ ID NOs: 79 and 89, a higher exposureand reduced clearance was observed for the serum albumin binder of SEQID NO:15.

TABLE 4 Results from PK profiling in rats. PK parameters KD on ratV_(ss) Cl MRT Construct SA (M) (mL) (mL/h) (h) T_(1/2eff) (h) SEQ ID NO:82  2.0E−08 33 0.75 44.1 30.6 (invention) SEQ ID NO: 89 >1.0E−07 45.64.22 11.4 7.9 (reference) SEQ ID NO: 90 >1.0E−07 38.3 1.81 21.8 15(reference) KD on rat SA was determined in SPR. The constructs of SEQ IDNOs: 82 (invention) and 89 and 90 (reference) were injected at differentconcentrations on immobilized rat SA on a ProteOn instrument. Bindingand dissociation were analysed at pH 7.4. PK parameters were calculatedfrom non-compartmental analysis of blood concentration over time aftersingle i.v. injection of 20 μg of the Nanobodies. V_(ss): volume ofdistribution at steady state; Cl: clearance; MRT: Mean Residence Time;T_(1/2eff): effective half-life calculated from MRT (T_(1/2eff) =ln2*MRT).

Example 6: In Vivo Safety of the Serum Albumin Binder of SEQ ID NO:15 inRat

Albumin is a carrier protein for many natural ligands, such asbilirubin, lipids, ions, sugars, metabolites. To use the serum albuminbinder of SEQ ID NO:15 for half-life extension of therapeutic compounds,it should not displace binding of natural ligands. This was assessed ina safety study in Crl:CD(SD) rats. Animals were injected i.v. with 100mg/kg of the construct of SEQ ID NO: 82 or vehicle (D-PBS) on day 1, 4and 7. Blood was collected on day 4, 7 and 12 and clinical parameterswere measured. The compound of the invention was well tolerated and didnot result in any adverse clinical observation, food consumption, bodyweight, or clinical chemistry changes.

Example 7: Crystal Structure

Crystals of human serum albumin in complex with the serum albumin binderof SEQ ID NO:15 were flash-frozen and measured at a temperature of 100K. Diffraction data for the co-crystallized complex were collected atthe SWISS LIGHT SOURCE (Villigen, Switzerland). Data collection andprocessing statistics are summarized FIGS. 7 and 8 .

The resulting electron density maps reveal that the crystals contain oneHSA:the serum albumin binder of SEQ ID NO:15 complex in the asymmetricunit and show the unambiguous binding mode for the serum albumin binderof SEQ ID NO:15, binding to domain II of HSA. A structural model wasconstructed and refined to a final resolution of 2.80 Å. The modelcomprises residues Glu1 to Ser123 of the serum albumin binder of SEQ IDNO:15 and Lys4 to Leu583 of HSA. The main residues that were found to beinvolved in the interaction of the serum albumin binder of SEQ ID NO:15with HSA are listed in FIG. 10 (see also FIG. 11 ).

1.-16. (canceled)
 17. An immunoglobulin single variable domain (ISVD) capable of binding to serum albumin that comprises: (i) a CDR1 (according to Abm) that is the amino acid sequence GLTFSSYAMG (SEQ ID NO: 12); (ii) a CDR2 (according to Abm) comprising: an R at position 52a according to Kabat numbering; a S at position 52 according to Kabat numbering; a G at position 55 according to Kabat numbering; a Y at position 56 according to Kabat numbering; a Y at position 58 according to Kabat numbering; and (iii) a CDR3 (according to Abm) comprising: a Y at position 97 according to Kabat numbering; a W at position 98 according to Kabat numbering; an A at position 99 according to Kabat numbering; a T at position 100 according to Kabat numbering; a S at position 100b according to Kabat numbering; an E at position 100c according to Kabat numbering; and a Y at position 100d according to Kabat numbering; optionally wherein the serum albumin is human serum albumin.
 18. The ISVD according to claim 17, further comprising a K at position 64 according to Kabat numbering.
 19. The ISVD according to claim 17, that can bind to human serum albumin with an affinity better than 100 nM or better than 50 nM, as determined by surface plasmon resonance.
 20. The ISVD according to claim 17, that is a heavy-chain immunoglobulin single variable domain.
 21. The ISVD according to claim 17, that is a VHH, a humanized VHH or a camelized VH, optionally wherein the camelized VH is a camelized human VH.
 22. The ISVD according to claim 17, wherein the ISVD has a serum half-life in human (expressed as t½ beta) that is more than 6 hours, 12 hours, 24 hours, or 72 hours.
 23. The ISVD according to claim 17, that has: a degree of sequence identity with the sequence of SEQ ID NO: 15 of at least 85%, at least 90%, or at least 95%; or that has: no more than 7, no more than 5, no more than 3, no more than 2, or no more than 1 amino acid differences with the sequence of SEQ ID NO: 15; wherein the CDRs are not taken into account for determining the degree of sequence identity or the amino acid differences.
 24. The ISVD according to claim 17, that is a VHH and that contains, compared to the sequence of SEQ ID NO:15, one or more humanizing substitutions.
 25. The ISVD according to claim 17, wherein the ISVD cross-blocks the binding of the amino acid sequence of SEQ ID NO: 15 to human serum albumin.
 26. An immunoglobulin single variable (ISVD) domain capable of binding to serum albumin and that is chosen from SEQ ID NOs: 15 to 77, optionally wherein the serum albumin is human serum albumin.
 27. A polypeptide that comprises at least one ISVD according to claim
 17. 28. The polypeptide according to claim 27, that is a fusion protein.
 29. The polypeptide according to claim 27, wherein: the polypeptide further comprises a C-terminal ISVD, wherein the C-terminal ISVD polypeptide comprises a C-terminal extension (X)n, wherein each X is an amino acid residue that is independently chosen from naturally occurring amino acid residues, and wherein n is 1-10; and/or the polypeptide further comprises a N-terminal ISVD, wherein the N-terminal ISVD polypeptide comprises a D at position
 1. 30. The polypeptide according to claim 27, that has a serum half-life in human (expressed as t½ beta) that is more than 6 hours, more than 12 hours, more than 24 hours, or more than 72 hours.
 31. The polypeptide according to claim 27, that comprises at least one therapeutic moiety or entity.
 32. The polypeptide according to claim 31, that has a serum half-life in human (expressed as t½ beta) that is more than 6 hours, more than 12 hours, more than 24 hours, or more than 72 hours.
 33. A pharmaceutical composition comprising a polypeptide according to claim
 27. 34. A nucleic acid that encodes an ISVD according to claim
 17. 35. An expression vector that comprises the nucleic acid of claim
 34. 36. A host cell that comprises an expression vector according to claim
 35. 37. A method for preparing a polypeptide comprising cultivating or maintaining a host cell according to claim 36 under conditions such that said host cell produces or expresses the polypeptide, and optionally further comprising isolating the polypeptide. 